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Capture, recapture, and trapping of molecules with a nanopore

a nanopore and nanopore technology, applied in the field of detection and characterization of molecules, can solve the problems of inability to achieve in-depth characterization of single molecules with a nanopore, either alone or as part of a more complicated device, and the inability to capture dna by a solid-state nanopore and translocation through the nanopor

Active Publication Date: 2011-08-30
PRESIDENT & FELLOWS OF HARVARD COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0007]With this system, a molecular species, such as a molecule or component of a molecule, can be captured, recaptured, and analyzed with regard to the conditions of the nanopore and conditions of the first and second reservoirs. As explained in detail below, the invention thereby enables a wide range of experiments and analyses that elucidate the nature of molecular behavior. The molecular capture and recapture control system provides knowledge of a molecule's position at a nanopore at both ends of a measured time interval, and provides knowledge of the forces applied to the molecule during that time interval, enabling an evaluation of the molecule's path in solution. The dynamics of a molecule reaching a nanopore can therefore be correlated with the dynamics of a molecule entering a nanopore, and each activity can be studied individually.

Problems solved by technology

Despite intense research interest, such DNA capture by a solid state nanopore and translocation through the nanopore is currently not well understood.
In-depth characterization of single molecules with a nanopore, either alone or as part of a more complicated device, cannot be accomplished until a more full understanding and the control of the dynamics of a molecule's interaction with a nanopore are achieved.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example i

[0103]Nanopores of about 6 nm in diameter were fabricated in a ˜20 nm thick SiN membrane using a condensed transmission electron microscopy (TEM) beam. The unsupported area of the membrane had lateral dimensions greater than 20 microns, whereby the nanopore could effectively be represented as a hole in an infinite electrically insulating sheet. To reduce the capacitance of the system the SiN membrane was supported on a 2 μm-thick silicon dioxide layer, which was provided on a 3 mm silicon wafer having a pyramidal pit fabricated by standard MEMS bulk micromachining. With this configuration, the total capacitance of the silicon wafer, nanopore reservoir flow cell arrangement, and fluid inputs was measured to be 13 pF.

[0104]The membrane-nanopore configuration provided on the silicon wafer was assembled in a PEEK flow cell with PDMS gaskets. After assembly, the wafer configuration, the holder, and the gaskets were oxygen plasma cleaned for 60 s at 100 W and 500 mT. Immediately after the...

example ii

[0127]The molecular capture-recapture system of Example I was modified to operate as a single-molecule spatial trap. A dilute concentration, 12 ng / μL, of a mixture of 5.4 kbp and 10 kbp DNA molecules was employed in the solution. This mixture of differing molecules was employed to enable the detection of a substitution of one molecule for another in the trap; if a second molecule were to displace the trapped molecule, there is a 50% chance that the detected molecular length would change, given the solution mixture. The relatively low concentration of molecules was employed here to decrease the probability that a second molecule from the cis reservoir would be close enough to be captured and replace an initially-trapped molecule. Also, at this concentration, under forward-translocation voltage bias polarity, new molecules arrived at the nanopore at a rate of under 0.4 Hz. Under reverse-translocation voltage bias polarity, the background arrival rate was an order of magnitude less. It...

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Abstract

In a molecular analysis system, there is provided a structure including a nanopore and first and second fluidic reservoirs. The two reservoirs are fluidically connected via the nanopore. A detector is connected to detect molecular species translocation of the nanopore, from one of the two fluidic reservoirs to the other of the two fluidic reservoirs. A controller is connected to generate a control signal to produce conditions at the nanopore to induce the molecular species to re-translocate the nanopore at least once after translocating the nanopore. This enables a method for molecular analysis in which a molecular species is translocated a plurality of times through a nanopore in a structure between two fluidic reservoirs separated by the structure.

Description

RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application No. 60 / 997,322, filed Oct. 2, 2007, the entirety of which is hereby incorporated by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH[0002]This invention was made with Government support under Contract No. 5RO0HG003703, awarded by NIH. The Government has certain rights in the invention.BACKGROUND OF INVENTION[0003]This invention relates generally to the detection and characterization of molecules, and more particularly relates to nanopore device configurations and corresponding techniques for the characterization of molecules.[0004]The detection, analysis, and quantification of molecules, and particularly biological molecules, has become important for a wide range of applications, e.g., in the areas of healthcare and the life sciences. Of particular interest is an ability to carry out single molecule sensing. The development of solid state nanopores has shown great potential for th...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C12Q1/68C12M1/00G01N15/06C07H21/04
Inventor GERSHOW, MARC H.GOLOVCHENKO, JENE A.BRANTON, DANIEL
Owner PRESIDENT & FELLOWS OF HARVARD COLLEGE